J. Sci. Food Agric.

1979, 30, 424-428

Hibiscus sabdarifla Seed Oil: A Re-investigationa

Moghis U. Ahmad, Syed K. Husain, Ishtiaque Ahmad and Sheikh M. Osman
Department of Chemistry, Aligurh Muslim University, AIigurh--702001, India
(Manuscript received I August 1978)

Due to controversial reports on the component acids of Hibiscus sabdarifa seed

fat and in anticipation of the co-occurrence of cyclopropene and epoxy acids in
the seed oils of Malvaceae (especially in the genus Hibiscus)5,6 it was considered
worthwhile to re-examine the seed oil of H . sabdarifla. Seed oil of H . sabduriflu
(Malvaceae) contains myristic (2.1 %), palmitic (35.2 %), palmitoleic (2.0%), stearic
(3.473, oleic (34.0 %), linoleic (14.6%) and three unusual HBr-reacting fatty acids.
These acids are cis-12,13-epoxy-cis-9-octadecenoic(12,13-epoxyoleic),4.5 %; sterculic,
2.9%; and malvalic, 1.3 %. Acetolysis of epoxide in the presence of cyclopropenes
was effected by room temperature treatment with acetic acid-lO% sulphuric acid.
Seed oils of Vernonia anthelmintica and Sterculia foetida were used as reference
standards.

1. Introduction
The seed fat of Hibiscus sabdarifa, commonly called roselle (Hindi; Patwa, La1 ambari)' has been
examined by several groups of worker^,^-^ and it is agreed that it contains an HBr-reacting acid.
Subbaram et u1.2 reported the presence of epoxyoleic acid (6%) in addition to the conventional
fatty acids, using the reversed-phase partition chromatography (r.p.p.c.) technique. Cornelius
et al.3 characterised the unusual component as sterculic acid ( 5 % ) on the basis of the Halphen
colour test and HBr-titration of the oil. Recently, Mohiuddin and Zaidi4 have determined the
fatty acid composition by gas-liquid chromatography (g.1.c.) of the methyl esters and the oxirane
oxygen was determined from the HBr-titration of the oil. These authors reported the component
acids as epoxyoleic (3.3 "/,), stearic, oleic, and linoleic acids. The complete absence of palmitic acid,
which appears to be quite unusual, has also been noted by them. The present work was under-
taken to characterise and estimate the individual HBr-reacting (cyclopropene and/or epoxy) acids
present in this seed oil.

2. Experiments and results

2.1. Materials and methods
Melting points were observed on Kofler apparatus and are uncorrected. Infrared (ix.) spectra were
obtained with a Perkin-Elmer 621 spectrophotometer as liquid film or as 1% solutions in carbon
tetrachloride. Ultraviolet (u.v.) spectra were determined with a Beckman DK-2A spectrophoto-
meter in methanol.
Analytical thin-layer chromatography (t.1.c.) was performed on plates coated with 0.25 or 0.1
mm layers of silica gel C or 20% silver nitrate-impregnated silica gel G with 20 or 30% ether in
hexane as the solvent. For preparative t.l.c., layers 1 .O mm thick were used. The preparative plates
were impregnated with dichlorofluorescein as an internal indicator and bands were visualised under
U.V. light. The reversed-phase t.1.c. was performed on dried coated plates impregnated with silicone
oil (E. Merck, A. C . Darmstadt). Acetonitrile-acetic acid-water (70: 10:20,by volume) was used
a Presented at the 33rd Annual Convention and Symposium of Oil Tech. Assn of India, Calcutta, February 1978.
0022-5142/79/04OCr0424 $02.00 01979 Society of Chemical Industry
424
Hibiscus sabdariflu seed oil 425

as the developing solvent. Dihydroxy acid methyl esters were analysed on silica gel G impregnated
with boric acid7 and developed with hexane-ether-acetic acid (60:40: 1, by volume). The spots on
all analytical t.1.c. plates were visualised by charring the plates at 120°Cafter they had been sprayed
with a 20% aqueous solution of perchloric acid. Triglycerides containing the epoxy group were
revealed by the on-the-plate test with picric acid.8
G.1.c. analysis was carried out with a Perkin-Elmer Model 154 instrument equipped with a
thermal conductivity detector, using a stainless steel packed column (2 m x Q in) coated with diethy-
lene glycol succinate (DEGS, 15 % on chromosorb W, 45-60 mesh). The separations were carried
out isothermally at 200"C, chart speed 30 in h-l, with a hydrogen flow of 70 ml min.-1 Peak
areas were calculated by triangulation.

2.2. Preliminary analysis of oil

Seeds were ground and analysed for oil content by overnight extraction with petroleum ether
(b.p. 40-60°C) in a Soxhlet extractor. Solvent was removed in vacuo at 40°C, and the oil was
titrated with HBr according to the procedure of Harris et al.9 at two different temperatures (3 and
55°C) and the results are expressed as hydrogen bromide equivalent (HBE). Titration results
indicated that the oil contained 8.6 % of HBr-reactive materials. The oil also responded to the
Halphen test,lO indicating the presence of cyclopropenoid material. The U.V. spectrum of the oil
showed no conjugation in the component acids of the triglycerides. The analytical values of oil
and seeds were determined according to the procedures recommended by the AOCS methods11
and the data are summarised in Table 1.

Table 1. Analytical data o n H. snbdariffa seeds

and oil

Seeds
Oil content (%) 16.0
Unsaponifiable content (%) 0.9
Protein content (%) 32.8
Moisture (%) 8.0

Seed oil
Iodine value (Wijs) 69.0
Saponification value 206.0
Refractive index, nD40 1.4715
Oxirane oxygen (%) 0.23
Halphen test Positive
HBE 3°C 4.5n
55°C 4.lb

QExpressed as % epoxyoleic.
bExpressed as % cyclopropene (sterculicl-
malvalic).

Analysis of oil by t.1.c. revealed three spots, RF 0.49, 0.42, and 0.34. The spot (RF 0.42) gave a
positive picric acid t.1.c. test,8 indicating the presence of epoxy acid as one of the HBr-reactive
materials.

2.3. Acetolysis of epoxide

A portion (20 g) of oil (HBE 8.6) was stirred overnight at room temperature with 80 ml of 10%
sulphuric acid in 200 ml of acetic acid, as described by Wilson et a1.6 The mixture was diluted with
distilled water and extracted with ether. The combined ether extracts were washed with water,
dried over anhydrous sodium sulphate, and evaporated in a stream of nitrogen to yield a viscous
oil.
Saponification of the recovered oil was effected by stirring overnight with 0 . 8 ~alcoholic KOH
28*
426 M. U.Ahmad er al.

at room temperature. The unsaponifiables were removed and the mixed fatty acids were recovered
by the usual ether extraction method which on direct t.1.c. revealed two distinct spots. Separation
of these mixed fatty acids into oxygenated and non-oxygenated fractions was accomplished by
preparative t.1.c.

2.4. Oxygenated fatty acid

The crude oxygenated fatty acid concentrate (0.84 g) on successive crystallisations from acetone
and petroleum ether-ethyl ether (3: 1, by volume) afforded a crystalline product of m.p. 5455°C
(lit.12 m.p. 53-54°C) and with the same RF value by t.1.c. as threu-12,13-dihydroxyoleic acid pre-
pared from V. unthelminticu seed oil. Analysis: calculated for C18H3404; C, 68.78;H,10.82%.
Found: C, 68.67; H, 10.67%. An admixture with authentic threu-12,13-dihydroxyoleicacid
melted at 5455°C.The unsaturated dihydroxy acid consumed 0.98 mole equivalents of hydrogen
(Pt catalyst) to yield saturated dihydroxy acid which on crystallisation from ethyl acetate melted
at 96-97°C (lit.lz m.p. 95-96°C). No depression of melting point was observed on admixture with
authentic threo-12,13-dihydroxystearicacid. Analysis: calculated for C18H3604: C, 68.35; H,
11.39%. Found: C, 68.26; H, 11.34%. Comparison of the methyl esters of the unknown diols
with those of the standard diols by t.1.c. on boric acid-impregnated7 silica gel G also demonstrated
that both had the threo configuration.
The dihydroxy acids were oxidised by periodate-permanganate13 and the products identified
by g.1.c. after methylation. The unsaturated acid gave hexanoic and azelaic acids and the saturated
acid gave hexanoic and dodecanedioic acids.

2.5. Non-oxygenated fatty acids

The methyl esters of the non-oxygenated acids, prepared with ethereal diazomethane, were examined
qualitatively by various t.1.c. techniques using S. fuetida esters as the cyclopropene acid reference.
Direct t.1.c. showed only non-oxygenated acids. The reversed-phase t.1.c. revealed a spot near the
starting point corresponding to the spot exhibited by S. foetida esters. Clear spots of usual critical
pairs were also obtained. T.1.c. of these esters on silver nitrate-impregnated silica gel G showed
spots of saturates, monoene and diene.
G.1.c. of the silver nitrate-methanol treated methyl esters14 clearly established the presence of
malvalic and sterculic acids in this seed oil by a comparison of the relative retention times of the
derivatives of S.foetida esters. The total cyclopropene content (Table 2) agrees approximately with
the HBr-titration result (Table 1).

Table 2. Fatty acid components of H. sabdurifla seed oil (% total fatty acids)

Reference

Component acids Subbararn et a1.2 Cornelius er a1.3 Mohiuddin and Zaidi4 Present work

Myristic - Trace 2.1

Palmitic 18.6 19.0 35.2
Palrnitoleic - 1 .o - 2.0
Stearic 1.2 2.0 23.1 3.4
Oleic 35.8 27.0 29.2 34.0
Linoleic 38.4 46.0 44.4 14.6
12,13-Epoxyoleic 6.0 - 3.3 4.5
Sterculic 5.0 2.9
Malvalic - 1.3

3. Discussion
Freshly extracted oil of H . sabduriflu (HBE 8.6)responded to the Halphen test,lO thereby indicating
the presence of cyclopropenoid material. Analysis of oil by t.1.c. revealed three spots (RF0.49,
Hibiscus sabdariffa seed oil 427

0.42, and 0.34). The spot ( R F0.42) gave a positive picric acid t.1.c. test,* indicating the presence of
epoxy acid in the seed glycerides. Acetolysis of epoxide was effected without destruction of the
cyclopropene moiety by treatment with acetic acid-10 % sulphuric acid (5 :2, by volume) at room
temperature, following the procedure of Wilson et aZ.6 The acetolysed oil was saponified by stirring
overnight with 0 . 8 ~alcoholic KOH at room temperature. The liberated fatty acids were separated
into oxygenated and non-oxygenated fractions by preparative t.1.c. and examined separately for
the characterisation of the individual fatty acids.

3.1. Characterisation of epoxy fatty acid

The oil showed 0.23 % oxirane oxygen, equivalent to 4.5 % epoxyoleic acid. Acetylation of the oil,
followed by saponification and separation of the acids, gave thre0-12,13-dihydroxyoleic acid
(m.p. and mixed m.p. 54-55°C). The yield of unsaturated dihydroxy acid was 0.84 g from 20 g of
the oil, equivalent to 4.2% of the weight of the oil. Quantitative determination of the oxirane
content of the oil is in fair agreement with the amount of dihydroxyoleic acid isolated. The un-
saturated dihydroxy acid analysed correctly for C18H3404 and had an i.r. absorption at 3400-3500
cm-1 (hydroxyl). 1.r. showed no trans absorption. The unsaturated dihydroxy acid on hydrogena-
tion consumed 0.98 mole equivalents of hydrogen (one double bond) to yield threo-12,13-di-
hydroxystearic acid (m.p. and mixed m.p. 96-97°C). Comparison of the mobility of saturated and
unsaturated diols with those of the authentic samples on direct and boric acid t.l.c.7 also
demonstrated the identity of the structure.
Oxidative cleavage of the saturated and unsaturated dihydroxy acids showed the unsaturated
compound to be 12,13-dihydroxyoleic acid derived from 12,13-epoxyoleic acid. The dihydroxy
acid is the threo isomer and therefore the epoxide function has the cis configuration.

3.2. Characterisation of non-oxygenated fatty acids

The non-oxygenated methyl esters (positive Halphen test) showed the i.r. band at 1008 cm-l
(cyclopropene). The g.1.c. analysis clearly demonstrated the presence of malvalic (1.3 %) and
sterculic (2.9 %) acids, in addition to normal fatty acids. Qualitative results of direct, reversed-
phase, and silver ion t.1.c. supported the findings of g.1.c. analysis. Noticeable differences in the
content of palmitic and linoleic acids from those reported earlier may be attributed to the effect
of soil and climate.
Contrary to the suggestion of Mohiuddin and Zaidi,4 H . sabdarifa oil cannot be utilised for
edible purpose due to the carcinogenic properties of these HBr-reacting acids. The presence of
smaller amounts of these biologically active acids might adversely affect its stability and nutritional
properties.

Acknowledgements
The authors are grateful to Professor Wasiur Rahman for providing the necessary facilities; CSIR
and ICAR (Government of India) for financial assistance. This research was financed in part by a
grant from the USDA under PL-480.

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